Action of pure phospholipase A2 and phospholipase C on human erythrocytes and ghosts

Roelofsen, B.; Zwaal, R.F.A.; Comfurius, Paul; Woodward, C.B.; Deenen, L.L.M. Van

doi:10.1016/0005-2736(71)90024-1

Cited by 122 publications

(27 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This may be consistent with the function of the enzyme, since B. cereus hydrolyses only the hydrophilic part of phospholipids in membranes and liposomes and probably does not penetrate the erythrocyte membrane [50], or the platelet membrane [S]. Its ability to hydrolyse a specific substrate depends, however, on the chain length of the acyl substituents ( Fig.3 and [45]), as well as on micelle size, and the enzyme is known to attack even the innermost of concentrically arranged membrane vesicles in preparations of tissue thromboplastin [51].…”

Section: Discussionsupporting

confidence: 75%

“…Purified B. cereus phospholipase C completely degraded phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in human erythrocyte ghosts [50] and phosphatidylethanolamine and phosphatidylcholine in toad erythrocytes depleted of ATP were extensively hydrolysed [57]. The present preparations of phospholipase C degraded phosphatidylcholine, phosphatidylethanolamine and phosphatiydlserine (but not sphingomyelin) from sonicated human blood platelets [S] and 80-90% of the phospholipid in isolated, detergent-washed HeLa cell nuclei [58].…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Some Characteristics of Phospholipase C from Bacillus cereus

Otnaess

Little

Sletten

et al. 1977

European Journal of Biochemistry

View full text Add to dashboard Cite

1. The amino acid composition of purified Bacillus cereus phospholipase C is reported. The enzyme contains one methionine residue and two fragments are obtained after cyanogen bromide cleavage. The sequence of the amino-terminal fragment (25 residues) is reported.2. Antisera were raised against the enzyme and purified by affinity chromatography. The antisera were monospecific and gave one precipitation line with purified as well as with crude phospholipase C, showing that no antigenic contaminants were present in the purified preparations used as antigen. The antibodies were purified to the extent that about two molecules neutralized one enzyme molecule.3. The enzyme is quite resistant to denaturation by urea, sodium dodecyl sulphate or heat (in the presence of 1 mM Zn").4. Phospholipase C hydrolyses phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Under the conditions used phosphatidylglycerol, cardiolipin, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine and lysophosphatidyl ethanolamine were not substrates. Replacement of Zn2+ by Co2+ or Ni2+ or variation of pH (7.2-8.3), did not change the range of substrates. Phosphatidylcholine was the best substrate among the isolated phospholipids and dicaproylphosphatidylcholine was clearly a better substrate than dipalmitoylphosphatidylcholine.

show abstract

Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Some Characteristics of Phospholipase C from Bacillus cereus

Otnaess

Little

Sletten

et al. 1977

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Haemolysis of the cells (determined as described in Ref. 14) in all experiments never exceeded 2%. As can be seen in Fig.…”

Section: Discussionmentioning

confidence: 91%

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

Roelofsen

Trip

Verheij

et al. 1980

Biochimica et Biophysica Acta (BBA) - Biomembranes

Self Cite

View full text Add to dashboard Cite

Summary1. Cobra venom phospholipase A2 from three different sources has been fractionated into different isoenzymes by DEAE ion-exchange chromatography. 2. Treatment of intact human erythrocytes with the various isoenzymes revealed significant differences in the degree of phosphatidylcholine hydrolysis in those cells. 3. It is argued that the plateaus observed in dose-response curves for such treatments may be caused by an increase in lateral surface pressure within the outer half of the membrane due to the production of free fatty acids and lyso-compounds.Phospholipases A2 (EC 3.1.1.4) purified from snake venoms (in particular from Naja naja venom) have been widely used as tools to study the disposition of phospholipids in biological membranes (for a recent review see Ref. 1). These enzymes have been particularly useful in elucidating the localization of phospholipids in the erythrocyte membrane [2], and the results thus obtained for the localization of phosphatidylcholine in that mem. brane could be quantitatively confirmed by studies using phosphatidylcholine exchange protein [3,4]. The reason why phospholipases could successfully be applied became apparent only recently, when analyses involving 3'P-NMR showed that even after extensive phospholipase treatments of the membrane, the residual phospholipids and products of hydrolysis remain in a bilayer configuration [5]. Indeed, fatty acids and lysophosphatidylcholine associate to form bilayers [6]. While the reliability of the results and the conclusions drawn from the above studies have been questioned by Martin et

show abstract

“…Unless otherwise stated, the mixtures were incubated for I h at 37 °C with gentle stirring, followed by centrifugation at 3000 × 9 for 5 min. The supernatants were collected and percentage haemolysis was determined as described previously [24].…”

Section: Treatment Of Erythrocytes With Phospholipasesmentioning

confidence: 99%

“…The phospholipids were separated by two-dimensional thin-layer chromatography using the procedure of Broekhuyse [26], and determined as phosphorus after destruction with 70 ~o HC104 at 190 °C by a modification [27] of the procedure of Fiske and SubbaRow. Percentage degradation of the different phospholipid classes was calculated as described previously [2,24].…”

Section: Phospholipid Analysismentioning

confidence: 99%

Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases

Zwaal

Roelofsen

Comfurius

et al. 1975

Biochimica et Biophysica Acta (BBA) - Biomembranes

Self Cite

349

119

View full text Add to dashboard Cite

SUMMARY1. The action of eight purified phospholipases on intact human erythrocytes has been investigated. Four enzymes, e.g. phospholipases A2 from pancreas and Crotalus adamanteus, phospholipase C from Bacillus cereus, and phospholipase D from cabbage produce neither haemolysis nor hydrolysis of phospholipids in intact cells. On the other hand, both phospholipases A2 from bee venom and Naja naja cause a non-haemolytic breakdown of more than 50 ~ of the lecithin, while sphingomyelinase C from Staphylococcus aureus is able to produce a non-lytic degradation of more than 80 ~o of the sphingomyelin.2. Phospholipase C from Clostridium welchii appeared to be the only lipolytic enzyme tested, which produces haemolysis of human erythrocytes. Evidence is presented that the unique properties of the enzyme itself, rather than possible contaminations in the purified preparation, are responsible for the observed haemolytic effect.3. With non-sealed ghosts, all phospholipases produce essentially complete breakdown of those phospholipids which can be considered as proper substrates for the enzymes involved.4. Due to its absolute requirement for Ca 2 ÷, pancreatic phospholipase A2 can be trapped inside resealed ghosts in the presence of EDTA, without producing phospholipid breakdown during the resealing procedure. Subsequent addition of Ca 2 ÷ stimulates phospholipase A z activity at the inside of the resealed cell, eventually leading to lysis. Before lysis occurs, however, 25 ~o of the lecithin, half of the phosphatidylethanolamine and some 65 ~ of the phosphatidylserine can be hydrolysed. This observation is explained in relation to an asymmetric phospholipid distribution in red cell membranes.Phospholipases used: phospholipase A2 (phosphatide acyl-hydrolyse, EC 3.1.1.4) from pig pancreas, bee venom, Naja naja, and Crotalus adamanteus, respectively. Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus and Clostridium welchii. Sphingomyelinase C (sphingomyelin cholinephosphohydrolase) from Staphylococcus aureus. Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) from Savoy cabbage. 84 5. It is concluded that the ability of the various phospholipases to attack the red cell membrane is dependent upon: (i) substrate specificity of the phospholipases, (ii) sidedness of the phospholipids when only one side of the membrane is exposed to phospholipase action, and (iii) compression state of the membranous lipid layer. The latter point is dealt with in more detail in the accompanying paper (Demel, R.

show abstract

Action of pure phospholipase A2 and phospholipase C on human erythrocytes and ghosts

Cited by 122 publications

References 15 publications

Some Characteristics of Phospholipase C from Bacillus cereus

Some Characteristics of Phospholipase C from Bacillus cereus

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases

Contact Info

Product

Resources

About